Cryptococcus neoformans causes a life threatening meningoencephalitis and despite treatments with amphotericin B and newer triazoles the management of this infection remains problematic. The foundation for this competing renewal remains the identification and characterization of Cryptococcus neoformans virulence genes through genetic manipulations for development of new drug targets. Our primary hypothesis is that under certain environmental stresses, C. neoformans will adapt by expressing genes which are essential for its survival and growth. This concept of environmental stresses regulating virulence genes has been very effectively used in plant pathogens and pathogenic bacteria to elegantly elucidate molecular mechanisms of virulence. Recently, we have proven that C. neoformans can similarly act as a model system for this strategy. For instance. we have discovered that C. neoformans uses the calcineurin A (CNA1) gene in the signaling pathway for 37 degrees Centigrade, Ph, Pco2 growth and virulence. Similarly, an alpha- heterotrimeric g-subunit protein encoded by a C. neoformans gene (GPA1) is essential for the signaling of nutrient starvation, low glucose, and iron deprivation for mating, capsule synthesis, melanin production and virulence. Furthermore, through several methods including cDNA library subtraction techniques, differential display RT-PCR, and in vivo expression technology with green fluorescent protein, we have now identified a series of regulated genes both to certain in vitro conditions and under an in vivo environment within the central nervous system of an immunosuppressed rabbit. In this competing renewal our focus continues to be a functional genomics approach which integrates these indirect methods of gene isolation by their regulatory status in the host and then characterization of these genes by examining site-directed gene knock-out mutants. Furthermore, our strategy includes both the identification and regulated genes by the host environment but also to use these genes in an attempt to identify potential central regulatory genes through one- and two-hybrid screens. It is our hypothesis that beginning to understand the genetic regulation of C. neoformans during infection through this proposal will be a powerful tool to find targets to interrupt pathogenesis.